This invention relates to column chromatography, and in particular to columns designed to separate components of whole blood.
Chromatography in general and high-performance liquid chromatography (HPLC) in particular are widely used for analytical and preparative separations of mixtures of chemical species. The columns used for HPLC are typically used for a series of successive analyses, particularly in automated chromatographic systems, and in general where the repeated use of a single column permits direct sample-to-sample comparisons without the error that is introduced by cleaning the separatory resin between injections or by exchanging one column for a fresh column of the same composition. Solid debris often enters these columns, having been entrained by the sample or the carrier fluid, and accumulates in the column, obstructing the free flow of fluid and causing back pressure to rise. The debris arises from several sources. Whole blood, for example, either diluted or undiluted, contains cell wall fragments and other insoluble matter. Included among this insoluble matter are insoluble proteins as well as soluble proteins that agglomerate and/or precipitate. Another source is the chromatographic instrument itself. Components of the instrument such as valves and pump seals tend to disintegrate and release particulate matter into the carrier fluid.
Debris from all of these sources tend to clog the column, inhibiting the free flow of liquid through the column and causing the back pressure to rise. Metal frits are usually present in the column to hold the resin, and these frits act as filters for the solid debris, but the frits themselves become clogged with the debris, again causing the column back pressure to rise. One of the sites where flow blockage occurs is the interface between the frit and the resin, since the contact between the frit and the resin reduces the sizes of the interstitial spaces in the interfacial region, rendering them narrower than those of either the frit or the resin themselves. The spaces at the interfacial region are small enough to block even the passage of very small proteinaceous material. This not only causes back pressure to rise but also interferes with the separation when these proteins are among the components sought to be identified and/or quantified.
Some of the debris can be removed by filtration or centrifugation that is off-line (i.e., before injection into any continuous-flow column system). This is a time-consuming step, however, and provides an added source of operator error. For on-line systems (i.e., automated, continuous-flow systems), guard columns and prefilters that are not in direct contact with the resin have been included, but for the reasons set forth in the preceding paragraph, back pressure still rises with repeated injections. The rising back pressure typically requires that the guard columns be replaced after 50 to 200 injections, depending on the application.